DNA molecules encoding cartilage-derived morphogenetic proteins

ABSTRACT

Nucleotide and amino acid sequences of cartilage-derived morphogenetic proteins (CDMP-1 and CDMP-2) from human and bovine cartilage extracts. These proteins exhibit chondrogenic activity and can be used to repair cartilage defects in a mammal.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of cartilageand bone development. More specifically, the invention relates tocartilage-derived morphogenetic proteins that stimulate development andrepair of cartilage in vivo.

BACKGROUND OF THE INVENTION

[0002] Bone morphogenetic proteins (BMPs) are members of the TGF-βsuperfamily that can induce endochondral bone formation in adultanimals. This superfamily includes a large group of structurally relatedsignaling proteins that are secreted as dimers and then cleaved toresult in biologically active carboxy terminal domains of the proteins.These bioactive proteins are characterized by 7 highly conservedcysteine residues. Interestingly, these proteins have different roles atvarious stages of embryogenesis and in adult animals. Recombinant BMPsare now available and have been shown to induce endochondral boneformation when assayed in vivo.

[0003] Indeed, the initial discovery of the BMPs was facilitated by suchin vivo assays for cartilage and bone development. These assays werebased on the observation that bone development could be initiated bysubcutaneous or intramuscular implantation of compositions comprising anextract of demineralized bone and residual bone powder. The novelproteins identified in the extracts were termed “bone morphogeneticproteins.” These proteins were subsequently classified as members of theTGF-β superfamily by virtue of amino acid sequence relatedness.Screening of genomic and cDNA libraries led to the isolation ofpolynucleotides encoding BMP-2, -3, -4, -5, -6 and -7.

[0004] One deficiency of the bone induction assay regards its inabilityto distinguish the physiological roles of different BMP family members.The cartilage and bone inducing activity of the BMPs is remarkablebecause the normal stages of endochondral bone formation that occurduring ontogeny are recapitulated in the adult animal. These stagesinclude mesenchymal condensation, cartilage and bone and bone marrowformation and eventual mineralization to produce mature bone.

[0005] Several observations suggest that BMPs have wide-rangingextraskeletal roles in development. First, localization studies in bothhuman and mouse tissues have demonstrated high levels of mRNA expressionand protein synthesis for various BMPs in kidney (BMPs -3, -4, -7), lung(BMPs -3, -4, -5, -6), small intestine (BMPs -3, -4, -7), heart (BMPs-2, -4, -6, -7), limb bud (BMPs -2, -4, -5, -7) and teeth (BMPs -3, -4,-7). Second, several members of the family, including BMP-4 and -7, arekey molecules in epithelial-mesenchymal interactions, for instanceduring odontogenesis. Third, BMP-2 and BMP-4 are involved in thesignaling pathway that controls patterning in the developing chick limband BMP-4 is a ventralizing factor in early Xenopus development. Fourth,Drosophila homologs of the BMPs, the decapentaplegic (dpp) and 60 A geneproducts, have the capacity to induce bone in mammals whereas humanBMP-4 confers normal embryonic dorso-ventral patterning in Drosophilatransformants defective in dpp expression. Thus, the BMPs are nowappreciated as pleiotropic cytokines.

[0006] Interestingly, none of the known BMPs are strongly expressed inthe chondroblasts and chondrocytes of the cartilage core of developinglong bones. The hypertrophic chondrocytes, where both Vgr-1 (BMP-6,(Lyons et al., Development 109:833 (1990)) and OP-1 (BMP-7)(Vukicevic etal., Biochem. Biophys. Res. Commun. 198:693 (1994)) have been found areexceptions in this regard.

SUMMARY OF THE INVENTION

[0007] One aspect of the present invention is a purified cartilageextract that stimulates local cartilage formation when combined with amatrix and implanted into a mammal. This extract can conveniently beproduced by a method which includes the steps of: obtaining cartilagetissue; homogenizing the cartilage tissue in the presence of chaotropicagents under conditions that permit separation of proteins fromproteoglycans; separating the proteins from the proteoglycans and thenobtaining the proteins. The step for separating the proteins from theproteoglycans can be carried out using a sepharose column. The extractcan also be obtained by additionally including the steps of separatingthe proteins on a molecular sieve and then collecting the proteinshaving molecular weights in the 30 kDa to 60 kDa size range. Articularcartilage or epiphyseal cartilage can be used in the preparation of thispurified extract.

[0008] A second aspect of the present invention is a method of preparinga partially purified articular cartilage extract having chondrogenicactivity. This method includes the steps of first obtaining cartilagetissue; homogenizing the cartilage tissue in the presence of chaotropicagents under conditions that permit separation of proteins fromproteoglycans; separating the proteins from the proteoglycans andfinally obtaining the proteins. The separation of proteins andproteoglycans can be accomplished using a sepharose column. Inparticular, the step for separating proteins from proteoglycans caninclude isolating the proteins that bind heparin Sepharose in thepresence of 0.15 M NaCl but not in the presence of 1 M NaCl. Anadditional step in the purification procedure can include separating theproteins on a molecular sieve and then obtaining the proteins havingmolecular weights between 30 kDa and 60 kDa.

[0009] A third aspect of the present invention is an isolated DNAmolecule that encodes a protein having chondrogenic activity in vivo butsubstantially no osteogenic activity in vivo. More particularly, thisaspect of the invention regards a molecule having a nucleotide sequencethat can hybridize to a polynucleotide which has the nucleotide sequenceSEQ ID NO:11 or SEQ ID NO:12 at 55° C. with 0.4×SSC and 0.1% SDS. Theproteins encoded by such DNA molecules can have the amino acid sequencesof SEQ ID NO:13 or SEQ ID NO:14.

[0010] A forth aspect of the present invention is a recombinant proteinhaving chondrogenic activity in vivo but substantially no osteogenicactivity in vivo. This protein can have the amino acid sequence of SEQID NO:13 or SEQ ID NO:14.

[0011] A fith aspect of the present invention is a method of stimulatingcartilage formation in a mammal. This method includes the steps:supplying cartilage-derived morphogenetic proteins having in vivochondrogenic activity; mixing the partially purified proteins with amatrix to produce a product that facilitates administration of thetpartially purified proteins and implanting this mixture into the body ofmammal to stimulate cartilage formation at the site of implantation. Thepartially purified cartilage-derived morphogenetic proteins can beobtained from either articular cartilage or epiphyseal cartilage. Thematrix can also include non-cellular material. Viable chondroblast orchondrocytes can also be included in the mixture prior to implantation.The mixture can be implanted either subcutaneously or intramuscularly.

[0012] A sixth aspect of the present invention is a composition that canbe administered to a mammal for the purpose of stimulating chondrogenicactivity at the site of administration without substantially stimulatingosteogenic activity. This composition comprises at least onecartilage-derived morphogenetic protein and a matrix. Thecartilage-derived morphogenetic protein can be derived from an extractof either articular cartilage or epiphyseal cartilage. In anotherembodiment, the cartilage-derived morphogenetic protein is a recombinantprotein. This recombinant protein can have the amino acid sequence ofeither SEQ ID NO:13 or SEQ ID NO:14. The matrix used to create thecomposition can be either fibrin glue, freeze-dried cartilage, collagensor the guanidinium-insoluble collagenous residue of demineralized bone.Alternatively the matrix can be a non-resorbable matrix such astetracalcium phosphate or hydroxyapatite.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 presents the nucleotide and predicted amino acid sequenceencoded by the full length human CDMP-1 cDNA.

[0014]FIG. 2 presents the nucleotide and predicted amino acid sequenceencoded by the bovine CDMP-2.

[0015]FIG. 3 presents the genetic maps of chromosome 2 showing thelocalization of CDMP-1. The map on the right is based on the data fromtwo separate crosses.

[0016]FIG. 4 shows an alignment of segments from predicted CDMP aminoacid sequences in standard one letter code.

DETAILED DESCRIPTION OF THE INVENTION

[0017] We discovered that partially purified extracts of newborn calfarticular cartilage contained an activity that induced cartilageformation when implanted subcutaneously in rats. This biologicalactivity was reminiscent of that which characterized the BMPs.Degenerate oligonucleotide primer sets derived from the highly conservedcarboxy-terminal region of the BMP family were employed in reversetranscription-polymerase chain reactions (RT-PCR) using poly(A)⁺ RNAfrom articular cartilage as a template. These procedures allowed us todetermine which BMPs were expressed in chondrocytes.

[0018] Two novel members of the TGF-β superfamily were identified anddesignated Cartilage-Derived Morphogenetic Protein-1 (CDMP-1), and -2(CDMP-2). The C-terminal TGF-β domains of these proteins were 82%identical, thus defining a novel subfamily most closely related toBMP-5, BMP-6 and osteogenic protein-1. Northern analyses showed thatpostnatally both genes were predominantly expressed in cartilaginoustissues. In situ hybridization and immunostaining of sections from humanembryos showed that CDMP-1 was predominantly found at the stage ofprecartilaginous mesenchymal condensation and throughout thecartilaginous cores of the developing long bones. CDMP-2 expression wasrestricted to the hypertrophic chondrocytes of ossifying long bonecenters. Neither gene was detectable in the axial skeleton during humanembryonic development. The cartilage-specific localization pattern ofthese novel TGF-β superfamily members, which contrasts with the moreubiquitous presence of other BMP family members, suggested a role forthese proteins in chondrocyte differentiation and growth of long bones.

[0019] The discovery of a novel subfamily of cartilage derivedmorphogenetic proteins suggested the existence of morphogens thatprimarily functioned in the induction and maintenance (i.e., balancingcartilage and bone formation at articular surfaces) of cartilaginous andbony tissues. This subfamily may also include key molecules that governbone marrow differentiation.

[0020] The cartilage-derived morphogenetic proteins contained in thecartilage extract of the present invention, and the recombinant CDMP-1and CDMP-2 proteins described herein are contemplated for use in thetherapeutic induction and maintenance of cartilage. For example, localinjection of CDMPs as soluble agents is contemplated for the treatmentof subglottic stenosis, tracheomalacia, chondromalacia patellae andosteoarthritic disease. Other contemplated utilities include healing ofjoint surface lesions (e.g. temporomandibular joint lesions or lesionsinduced posttraumatically or by osteochondritis) using biologicaldelivery systems such as fibrin glue, freeze-dried cartilage grafts, andcollagens mixed with CDMPs and locally applied to fill the lesion. Suchmixtures can also be enriched with viable cartilage progenitor cells,chondroblasts or chondrocytes. We also contemplate repair orreconstruction of cartilaginous tissues using resorbable ornon-resorbable matrices (tetracalcium phosphate, hydroxyapatite) orbiodegradable polymers (PLG, polylactic acid/polyglycolic acid) coatedor mixed with CDMPs. Such compositions may be used in maxilofacial andorthopedic reconstructive surgery. Finally, the CDMPs disclosed hereinhave utility as growth factors for cells of the chondrocyte lineage invitro. Cells expanded ex vivo can be implanted into an individual at asite where chondrogenesis is desired.

[0021] We also anticipate the polynucleotides disclosed herein will alsohave utility as diagnostic reagents for detecting genetic abnormalitiesassociated genes encoding CDMPs. Diagnostic testing could be performedprenatally using material obtained during amniocentesis. Any of severalgenetic screening procedures could be adapted for use with probesenabled by the present invention. These procedures include restrictionfragment length polymorphism (RFLP), ligase chain reaction (LCR) orpolymerase chain reaction (PCR).

[0022] We began our investigations by considering whether there weredifferences between the chondrogenic/osteogenic differentiation factorsthat characterized calcifying (epiphyseal, scapular cartilage) andnon-calcifying (articular, nasal septum) cartilage tissues. It had beenpreviously established that tail tendon, achilles tendon, cartilage andskin matrices were devoid of chondrogenic/osteogenic activity(originally described as “transforming potency”) as measured in an invivo subcutaneous implantation model in rats (Reddi A. H., 1976,“Collagen and Cell differentiation” in Biochemistry of Collagen, eds.Ramachandran G. N. and Reddi, A. H., pp449-478, Plenum Press, New Yorkand London.).

[0023] We confirmed the absence of chondrogenic or osteogenic activityin crude 4 M guanidine HCl (GdnHCl) extracts of cartilage matrices, butunexpectedly discovered in vivo chondrogenic activity in the 0.15 M NaCleluate of the cartilage extracts after ion exchange chromatography. Thedevelopment of a unique extraction procedure (1.2 M GdnHCl and 0.5%CHAPs) followed by a heparin Sepharose affinity chromatography stepconfirmed the presence of in vivo chondrogenic activity in cartilaginoustissues. This was especially true in bovine articular and epiphysealcartilage. When the bioactive heparin Sepharose eluates (1M NaCl eluate)were further purified using previously established procedures, molecularsieve chromatography and Con A affinity chromatography steps followed bySDS polyacrylamide gel electrophoresis and gel elution, chondrogenicactivity was established. Implantation of 0.5 to 1 μg gel elutedmaterial resulted in in vivo chondrogenesis. Surprisingly, and incontrast to the bone matrix purified activity, none of the peptidesequences that were found in tryptic digests of the highly purifiedcartilage extracts corresponded to any of the known BMPs. However, thebiological activity present in the extracts was reminiscent of BMP-likeactivity by virtue of its loss of activity upon reduction andalkylation, its affinity for heparin Sepharose and Con A.

[0024] Although other materials and methods similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are nowdescribed. General references for methods that can be used to performthe various nucleic acid manipulations and procedures described hereincan be found in Molecular Cloning: A Laboratory Manual (Sambrook et al.eds. Cold Spring Harbor Lab Publ. 1989) and Current Protocols inMolecular Biology (Ausubel et al. eds., Greene Publishing Associates andWiley-lnterscience 1987). The disclosures contained in these referencesare hereby incorporated by reference. A description of the experimentsand results that led to the creation of the present invention follows.

[0025] We initially discovered that an extract of cartilage possessed aunique chondrogenic activity. In particular, we discovered that newbornarticular cartilage contained chondrogenic activities when assayed inthe in vivo subcutaneous implantation model. Using a procedure adaptedfrom that used for the isolation of BMPs from demineralized bone matrix,we partially purified this activity and thereby provided evidence forthe presence of BMP-like molecules in cartilage.

[0026] Example 1 describes the biochemical methods used to characterizea chondrogenic activity present in bovine cartilage.

EXAMPLE 1 Characterization of Cartilage Derived Chondrogenic Activity

[0027] Articular (metatarsophalangeal joints), scapular and nasalcartilage (300 grams wet weight per tissue) were prepared from newborncalves. Epiphyseal cartilage was dissected from fetal bovine femurs (7-8months). The tissues were finely minced and homogenized with a Polytron(top speed, 2×30 seconds) in 20 volumes of 1.2 M GdnHCl, 0.5% CHAPS, 50mM Tris-HCl pH 7.2, containing protease inhibitors and extractedovernight at 4° C. as described by Luyten et al., in J. Biol. Chem.264:13377 (1989), which is hereby incorporated by reference. Thedisclosure of this article is hereby incorporated by reference. Sorgenteet al., (Biochem Biophys. Acta. 282:441 (1972)) disclosed theseprocedures extract >90% of the lower molecular weight matrix whileleaving most of the high molecular weight proteoglycans behind. Theextracts were concentrated and exchanged with 6 M urea by diafiltrationusing an Ultrasette™ (Filtron Technology Inc., MA) and loaded on a 0.5 Lheparin Sepharose (Pharmacia/LKB, NJ) column. Thereafter, the column waswashed with 5 bed volumes of 6 M urea, Tris HCl pH 7.4 with 0.15 M NaCl,and then eluted with 2 vol 1 M NaCl in the same buffer. Chondrogenicactivity was assayed by reconstituting a portion of the eluate with 25mg of guanidine-insoluble collagenous residue of demineralized rat bonematrix according to procedures described by Luyten et al., in J. Biol.Chem 264:13377 (1989). Implants were recovered after 10 days andalkaline phosphatase activity was measured as a biochemical indicator ofcartilage and/or bone formation. The specific activity was expressed asunits of alkaline phosphatase/mg of protein used for reconstitution inthe bioassay. Implants were also examined histologically for evidence ofcartilage formation using standard procedures known to those of ordinaryskill in the art.

[0028] Additional purification steps were also performed. The 1 M NaCleluate of articular cartilage, which contained biological activity, wasconcentrated by diafiltration and loaded onto a Sephacryl S-200 HR gelfiltration column (XK 50/100, Pharmacia/LKB, NJ). After molecular sievechromatography, the bioactive fractions were pooled and exchanged with50 mM Hepes, pH 7.4, containing 0.15 M NaCl, 10 mM MgSO₄, 1 mM CaCl₂ and0.1% (w/v) CHAPS using Macrosep™ concentrators (Filtron Technology Inc.,Northborough, Mass.). The equilibrated sample was mixed with 1 ml Con ASepharose (Pharmacia-LKB) previously washed with 20 volumes of the samebuffer according to the procedure described by Paralkar et al., inBiochem. Biophys. Res. Comm. 131:37 (1989). After overnight incubationon an orbital shaker at 4° C., the slurry was packed into a disposable0.7 cm ID Bio-Rad column and washed with 20 volumes of the Hepes bufferto remove unbound proteins. Bound proteins were eluted with 20 volumesof the same buffer containing 500 mM methyl-D-mannopyronaside. Theeluate was concentrated to 200 μl using Macrosep™ concentrators.Macromolecules were then precipitated overnight with 9 volumes ofabsolute ethanol at 4° C. The precipitate was redissolved in 1 ml 6 Murea, Tris HCl pH 7.4. The bioactive bound protein was then mixed with 2X Laemmli sample buffer (without reducing agents) and electrophoresed ona 12% preparative SDS/polyacrylamide gel. Gel elution of the separatedprotein fractions and testing for biological activity was performed asdescribed by Luyten et al., in J. Biol. Chem. 264:13377 (1989). We alsoobserved that, after reduction with dithiothreitol and alkylation withiodoacetamide, substantially all of the cartilage-forming activitycontained in the protein sample was lost.

[0029] Results indicated that each of the crude extracts of thedifferent cartilaginous tissues (articular, nasal, scapular orepiphyseal) were inactive when tested directly in the in vivo cartilageand bone inducing assay. This finding confirmed previously describedresults published by Reddi in “Collagen and Cell differentiation” inBiochemistry of Collagen (eds. Ramachandran G. N. and Reddi, A. H.,pp449-478, Plenum Press, New York and London (1976)). However, afterheparin affinity chromatography (Sampath et al., Proc. Natl. Acad. Sci.U.S.A. 84:7109 (1987)), chondrogenic activity was recovered in the 1 MNaCl eluate from articular cartilage extracts. An additional molecularsieve chromatography step (S200) was required to recover chondrogenicactivity from epiphyseal cartilage extracts. Similar results wereobtained upon ion exchange chromatography using DEAE Sephadex (0.15 MNaCl eluate). Significantly, no activity was detected in the extracts ofthe other cartilaginous tissues.

[0030] The highest specific activity was obtained for material derivedfrom articular cartilage (1 U alkaline phosphatase/mg protein). Thismaterial was used for characterization of the bioactivity. Furtherpurification of the active fraction by molecular sieve chromatography onSephacryl S-200HR (specific activity 112 U/mg), and affinitychromatography on Concanavalin A (specific activity 480 U/mg),established the presence of cartilage and bone inducing activitycharacteristic of the members of the BMP family. Gel elution experimentswith the Con A bound bioactive fraction demonstrated that the activityresided between roughly 34 and 38 kDa (specific activity of the geleluted fractions was 2143 U/mg). We have also demonstrated that sizeseparation by molecular sieve chromatography can be used to purifybiological activity in the 30-60 kDa size range. In addition, loss ofactivity that was observed following reduction and alkylation suggestedthat the bioactivity was induced either by a known or a new member(s) ofthe BMP family.

[0031] Given the demonstration that cartilage contained a BMP-likeactivity, we proceeded to isolate polynucleotides encoding theresponsible proteins. Specifically, degenerate primers corresponding toconserved regions of known BMPs were designed. These primers were thenemployed to amplify polynucleotides using reverse transcribed mRNA fromarticular cartilage as a template. These procedures ultimately led tothe identification of two novel cDNAs, which we called CDMP-1 andCDMP-2.

[0032] Example 2 describes the methods used to amplify polynucleotidescorresponding to mRNAs that were expressed in cartilage tissue and thatexhibited at least weak sequence similarity to conserved regions of theBMP mRNAs.

EXAMPLE 2 PCR Amplification of cDNAs Encoding Cartilage-DerivedMorphogenetic Proteins

[0033] Total RNA from bovine articular chondrocytes (metatarsophalangealjoints) was extracted using a modified acid guanidine-phenol-chloroformmethod described by Chomczynski et al., in Anal. Biochem. 162:156 (1987)and by Luyten et al., in Exp. Cell. Res. 210:224 (1994). Poly(A)⁺ RNAwas isolated using magnetic beads (PolyATract™, Promega, Madison, Wis.).Four degenerate oligonucleotide primers corresponding to highlyconversed motifs in the C-terminal region of the BMPs were used; S1:5′-GGITGG(C/A)AIGA(C/T)TGGAT(A/C/T)(A/G)TIGC(A/C/G/T)CC-3′ (SEQ ID NO:1)corresponding to amino acids [GW(Q/N)DWI(I/V)AP] (SEQ ID NO:2); S2:5′-GGITGG(A/T)(G/C)(I)GA(G/A)TGGAT(T/C/A)ATI(A/T)G(A/C/G/T)CC-3′ (SEQ IDNO:3) corresponding to amino acids [GWSEWIISP] (SEQ ID NO:4); AS1:5′-A(A/G)A/G)GT(C/T)TG(A/C/G/T)AC(A/G)AT(A/G)GC(A/G)TG(A/G)T -3′ (SEQ IDNO:5) corresponding to amino acids [NHAIVQTL] (SEQ ID NO:6);AS2:5′-CAI(C/G)C(A/G)CAI(G/C)(A/C/T)I(C/T)(C/G/T)IACIA(C/T)CAT-3′ (SEQID NO:7) corresponding to amino acids [M(V/I)V(E/R)(G/S/A)C(G/A)C] (SEQID NO:8). Nucleotides in parenthesis denote sites of degeneracy and Idenotes inosine. First strand cDNA synthesis was performed using 1 μgPoly(A)⁺ or 5 μg total RNA with oligo dT, random hexanucleotide primers,or the antisense degenerate primers, AS1 and AS2. Successful PCRamplifications were performed with the degenerate sense primers, S1 orS2 in combination with the AS 1 antisense primer were performed usingconditions described by Wharton et al., in Proc. Natl. Acad. Sci. U.S.A.88:9214 (1991). The reaction products were electrophoresed on 1.2%agarose gels, and DNA fragments of appropriate sizes were excised andpurified using the Magic PCR Prep DNA purification system (Promega,Madison, Wis.). Reamplification was performed with the same primers andeach PCR product was subcloned into the PCR II vector using the TACloning™ System (In Vitrogen Corporation, San Diego, Calif.). Results ofRT-PCR using poly(A)⁺ RNA isolated from newborn bovine articularcartilage as template and sets of degenerate oligonucleotide primers(S1/AS1 and S1/AS2) yielded amplification products of 120 bp and 280 bp.

[0034] Subcloned inserts were sequenced according to the dideoxy DNAsequencing method of Sanger et al., (Proc. Natl. Acad. Sci. U.S.A.74:5463 (1977)). Both DNA strands were sequenced using Sequenase Version2.0 DNA polymerase according to manufacturer's instructions (USB,Cleveland, Ohio) with at least two-fold redundancy. Confirmatory data inambiguous regions were obtained by automated thermal cycle sequencingwith an Applied Biosystems Model 370A sequencer and by using 7-deaza-GTP(USB, Cleveland, Ohio). The sequencing data were obtained fromrestriction fragments subcloned into pBluescript (Stratagene, La Jolla,Calif.) using either M13 forward and reverse primers or syntheticoligonucleotide primers.

[0035] The results from a computer-assisted search of the nucleic acidsequence databases indicated the cloned inserts encoded BMP-2, -6, BMP-7(OP-1), and several other BMP-like sequences. Identification of theselatter gene fragments led us to isolate larger cDNAs that included theentire protein coding region of the transcript. The availability of suchclones facilitated both a more precise analysis of the encoded BMP-likeprotein and permitted studies aimed at localizing the expression ofthese genes. Thus, cloned inserts having novel BMP-like sequences wereisolated, radiolabeled and used to screen both human and bovinearticular cartilage cDNA libraries.

[0036] Example 3 describes the methods used to isolate human and bovinecDNAs that corresponded to a segment of one of the BMP-like genesegments that were amplified from cartilage mRNA templates.

EXAMPLE 3 Library Screening

[0037] A 120 bp PCR fragment encoding part of the C-terminal domain ofnovel BMP like genes (dashed line, FIG. 1) was used to screen two cDNAlibraries. One library, from adolescent human articular cartilagepoly(A)⁺ RNA (kindly provided by Dr. Björn Olsen, Harvard, Boston,Mass.), was primed with oligo dT and constructed in the λgtl 1 vector.The other was a bovine oligo dT and random primed articular cartilagecDNA library constructed in the UNIZAP®XR vector (Stratagene, La Jolla,Calif.). Approximately 1×10⁶ plaques from each library were screened bystandard procedures. Hybridizations were performed for 20 hours at 42°C. in 6×SSC, 1×Denhardt's solution, 0.01% tRNA, 0.05% sodiumpyrophosphate and the membranes (DuPont 137 mm nylon membranes, NewEngland Nuclear, MA) were washed to final stringency of 6×SSC, 0.1% SDSat 55° C. for 20 minutes.

[0038] Thus, cloned inserts having novel BMP-like sequences wereisolated, radiolabeled and used to screen both human and bovinearticular cartilage cDNA libraries. Six clones were isolated from thehuman cDNA library. The sizes of the EcoRI inserts (2.1 kb) and theirrestriction maps were found to be identical for all six clones. Oneclone was used for nucleotide sequencing. An open reading frame encodinga BMP related protein, designated CDMP-1, was identified. It appearedthat the human cDNA clone lacked the coding region for the firstmethionine and signal peptide. The 5′ end of the human CDMP-1 wassubsequently obtained from a human genomic clone isolated from a libraryconstructed in the EMBL-3 vector (Clontech, Palo Alto, Calif.). The 5′end of human CDMP-1 contained a consensus translation initiationsequence disclosed by Kozak (J. Biol. Chem. 266:19867 (1991))immediately followed by a putative transmembrane signal sequencedescribed by Von Heijne (Nucl. Acids Res. 14:4683 (1986)). Thenucleotide sequence and the translation of the open reading frame ofCDMP-1 are presented in FIG. 1. As shown in the figure, the CDMP-1protein was predicted to have 500 amino acids, to consist of apro-region of 376 amino acids, a typical cleavage site(Arg-Xaa-Xaa-Arg/Ala) (SEQ ID NO:9), and a C-terminal domain of 120amino acids containing the seven highly conserved cysteinescharacteristic of the TGF-β gene family. A single N-linked glycosylationsite is located in the pro-region (marked by an asterisk in the figure).A putative signal peptide is underlined in bold. A termination codon(TGA) is shown in the 5′ untranslated region. The bold dashed underlineindicates the fragment obtained by RT-PCR that was subsequently used toscreen cDNA libraries. The 13 amino acid peptide used to raisepolyclonal antibodies in rabbits is underlined. A vertical arrowheadmarks the boundary between the sequence obtained from genomic DNA andcDNA.

[0039] Two clones with inserts of 2.8 kb were isolated from a bovinearticular cartilage cDNA library. Both clones were sequenced and theopen reading frame was found to encode another novel TGF-β relatedprotein, designated CDMP-2. The CDMP-2 cDNA and predicted proteinsequences are presented in FIG. 2. As shown in the figure, the openreading frame contained a putative proteolytic processing site (boxed),preceding a 120 amino acid mature C-terminal region containing sevenhighly conserved cysteines. The 5′ end with the first methionine andsignal peptide were missing. The product obtained by RT-PCR (bold dashedunderline) was used to screen a bovine cDNA articular cartilage library.The ApaI sites used to release a cDNA fragment for hybridizationexperiments are underlined. At the 5′ end, the pro-region lacked thefirst methionine and signal peptide. The mature C-terminal domain of 120amino acids showed 82% identity with CDMP-1.

[0040] Alignment of the carboxy terminal domains of CDMP-1 and -2 withother members of the BMP family revealed an amino acid identity of about50% with BMP-5, BMP-6 and OP-1 (BMP-7). These results suggested thatCDMP-1 and CDMP-2 are members of a new subfamily.

[0041] The amino acid sequence similarity between the human CDMP-1 andbovine CDMP-2 proteins prompted us to further investigate conservationof the CDMPs across different species. In particular, we employed a PCRamplification protocol to isolate CDMP cDNA sequences from a variety ofspecies. Based on alignments of the predicted proteins encoded by thesecDNAs, we identified a highly conserved amino acid sequence spanning 31residues. Only 5 amino acid positions within this sequence showedvariability. All remaining positions were identical for all isolates. Asdisclosed in the following Example, even the 5 variable positions showeda high degree of conservation. This structural conservation likelyrepresents a functional domain that is characteristic of the CDMP familyof proteins. Those of ordinary skill in the art will appreciate thatsuch extraordinary amino acid sequence conservation is indicative of afunctional domain. We therefore believe the consensus amino acidsequence presented in the following Example is critical to thebiological activity of the CDMPs.

[0042] Example 4 describes the procedures used to identify an amino acidconsensus sequence that characterizes the CDMPs from several differentspecies.

EXAMPLE 4 Identification of a Highly Conserved Consensus Sequence inCDMP Proteins

[0043] RNA isolated from chicken sternal cartilage, bovine articularcartilage and human articular cartilage was employed as the template inRT-PCR protocols using the primers S 1 and AS1 and procedures describedunder Example 2. Genomic DNA isolated from Xenopus and zebrafish wasalso used as the template for amplification of related gene sequences ina PCR protocol that employed the same primer sets. Amplified DNAfragments were subcloned according to standard procedures. The insertsfrom various isolates were sequenced by standard dideoxy chaintermination protocols. Aligned segments of the predicted proteinsencoded by the cloned cDNAs are presented in FIG. 4.

[0044] Results of the protein alignments clearly indicated that CDMPfamily members from several species shared a common amino acid sequencemotif in the region of the proteins encoded by the amplified cDNAsegments. Of the 31 amino acid positions presented in FIG. 4, all but 5were occupied by identical amino acid residues for all of the isolates.The variable amino acids were located at positions 3, 7, 11, 16 and 18.Position 3 was occupied either by I, M or V. Position 7 was occupied byeither D or E, both of which have acidic side groups. Position 11 wasoccupied by either Y, F or H. Position 16 was occupied by L or V, andposition 18 was occupied by D or E. The consensus deduced from thisalignment was:W-I-(I/M/V)-A-P-L-(D/E)-Y-E-A-(Y/F/H)-H-C-E-G-(L/V)-C-(D/E)-F-P-L-R-S-H-L-E-P-T-N-H-A(SEQ ID NO:15). This consensus sequence is slightly broader than the oneshown in FIG. 4, as it encompasses all the variations observed in thesequenced polynucleotides. The consensus sequence in the figureindicates predominating amino acids.

[0045] We believe that biologically active CDMPs will possess thishighly conserved amino acid sequence motif. Proteins having differentamino acids in the variable positions in the consensus will likelyrepresent novel family members having distinct functions. We alsobelieve that polynucleotide hybridization probes or PCR primers designedbased on this conserved protein motif can be used to isolate cDNAsencoding CDMP family members or related proteins.

[0046] Southern analyses were also carried out to investigate possiblesequence conservation across species and to localize the CDMP-1 gene toa particular chromosome.

[0047] Example 5 describes the Southern blotting protocols used todetect DNA sequences corresponding to the CDMP-1 cDNA.

EXAMPLE 5 Genetic Mapping of CDMP-1

[0048] Southern hybridization was performed using the evolutionaryrelatedness blot (Bios Laboratories, New Haven, Conn.) under conditionsrecommended by the manufacturer. The panel of EcoRI-digested genomicDNAs included human (homo sapiens), mouse (Mus musculus), chicken(Gallus domesticus), frog (Xenopus laevis), lobster (Homarusamericanus), mussel (Mytilus edulis), fish (Tautoga onitis), fruit fly(Drosophila melanogaster), nematode (Caenorhabditis elegans), yeast(Saccharomyces cerevisiae) and bacteria (E. coli). The 2.1 kb CDMP-1EcoRI fragment originally obtained from the cDNA library was used as aprobe, and the blot was washed to a final stringency of 0.4×SSC, 0.1%SDS, at 55° C.

[0049] Results from these Southern analyses using the original 2.1 kbhuman CDMP-1 cDNA probe (starting from amino acid position 40), showed5.9 and 2.6 kb bands in humans and strong hybridization in both mouseand chicken. Fainter bands were seen in fish, frog and lobster after 5days autoradiographic exposure. No hybridization was detected toDrosophila DNA.

[0050] The 2.1 kb ApaI fragment of CDMP-1 was used as a hybridizationprobe on Southern blots to type mouse genomic DNAs from two geneticcrosses: (NFS/N or C58/J×M. m. musculus)×M. m. musculus (see Joseph etal., Mol. Immunol. 30:733 (1990)) and (NFS/N×M. spretus)×M. spretus orC58/J (see Adamson et al., Virology 183:778 (1991)). DNAs from thesecrosses have been typed for over 650 markers including the Chr 2 markersSnap (synaptosomal associated protein 25), Psp (parotid secretoryprotein), Emv15 (ecotropic murine leukemia virus 15), Src (srconcogene), and Cd40 (cluster designation 40). Probes for these markersand restriction fragment length polymorphisms used to type these crosseshave been described by Joseph et al., in Mol. Immunol. 30:733 (1990) andby Grimaldi et al., in J. Immunol. 149:3921 (1992). Src was typed usinga mouse Src probe obtained from E. Rassart (U. Quebec, Montreal)following XbaI digestion in the musculus cross and BamHI digestion inthe spretus cross.

[0051] Results from Southern blotting with the 2.1 kb cDNA describedabove identified EcoRI fragments of 7.1 and 2.0 kb in M. m. spretus andM. m. musculus and 6.8 and 3.2 kb in NFS/N and C58/J.

[0052] Inheritance of the polymorphic fragments in the progeny of thetwo crosses used for mapping was compared with inheritance of over 650markers previously mapped to all 19 autosomes and the X chromosome. Thegene encoding CDMP-1 was found to be linked to markers on Chr 2 justproximal to Src. The closest linkage was observed with Psp and Emv15. Norecombination was observed between Cdmp1 and Psp in the 100 mice typedfor both markers indicating that these genes are within 3.0 cM at the95% confidence level. Similarly, the absence of recombination betweenGdf5 (Storm et al., Nature 368:639 (1994)) and Cdmp1 in 125 micesuggested these genes colocalized within 2.4 cM. This map locationsuggested close proximity to the brachypodism locus (bp). A genetic mapthat presents the localization of CDMP-1 on chromosome 2 is shown inFIG. 3. Recombination fractions are given to the right of each map ofthe diagram for each adjacent locus pair or cluster. Numbers inparenthesis represent the percent recombination and standard errorcalculated as described by Green in Genetics and Probability in AnimalBreeding Experiments, Oxford University Press, New York (1981). The mapon the left is an abbreviated version of the Chr 2 Committee Mapdisclosed by Siracusa et al., in Mammal Genome 4:S31 (1993), and showsthe map location of bp relative to the other markers typed in thecrosses used here.

[0053] The brachypodism (bp mice) disorder is characterized by adistinct shortening of the limbs without other tissue abnormalities. Thedefect has previously been attributed to lack of production of achondrogenic signal by mesenchymal cells at the time of chondrogenesis(Owens et al., Dev. Biol. 91:376 (1982)). During the course of ourinvestigation, an independent study by Storm et al. (Nature 368:639(1994)) described the isolation of the mouse CDMP-1 homolog, calledGdf-5, and established its linkage to the bracypodism (bp) mutation. Thetypes of mutations observed in bp mice were found to be effectivenull-mutations for the gene encoding Gdf-5/CDMP-1. The pattern ofexpression of CDMP-1 throughout the cartilaginous core observed duringhuman embryonic long bone development, coupled with the bp mutation inmice, indicated that its primary physiological role was most likely atthe stage of early chondrogenesis and chondrocyte differentiation in thedeveloping limb.

[0054] The foregoing results indicated the CDMP-1 and CDMP-2 cDNAs werenovel, exhibited moderate sequence conservation across species as judgedby evolutionary hybridization studies and that the CDMP-1 gene localizedto mouse chromosome 2. We proceeded to examine the pattern of CDMPexpression at the mRNA level.

[0055] Example 6 demonstrates the methods used to determine the patternof CDMP mRNA expression.

EXAMPLE 6 CDMPs are Predominantly Expressed in Cartilage DuringPostnatal Life

[0056] Equal amounts of poly(A)⁺ RNA (2 μg) from bovine criocoid andarticular cartilage were electrophoresed on 1.2% agarose-formaldehydegels and then transferred to Nytran membranes (Schleicher and Schuell,Kenne, N H) according to standard laboratory procedures. Multiple TissueNorthern blots were obtained from Clontech (Palo Alto, Calif.). Themembranes were prehybridized for 3 hours at 42° C. in hybridizationbuffer (5×SSPE, 5×Denhardt's solution, 50% formamide, 1% SDS and 100μg/ml freshly denatured salmon sperm DNA). Hybridizations with [³²P]dCTPlabeled probes, having specific activities of at least 1×10⁹ CPM/μg,were performed overnight under the same conditions as theprehybridization. Probes included the cDNA probe for humanglyceraldehyde-3-phosphate dehydrogenase (1.1 kb, G3PDH (Clontech, PaloAlto, Calif.), an ApaI fragment (bp 470-1155) of CDMP-1, and an ApaIfragment (bp 194-677) of CDMP-2. The CDMP-1 and CDMP-2 probes werechosen to avoid the highly conserved carboxy-terminal domain, therebyminimizing the potential for cross hybridization with other members ofthe gene family. Following hybridization, the filters were washed to afinal stringency of 55° C., 0.4×SSC, 0.1% SDS. The mRNA expressionlevels were quantified using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

[0057] Results from Northern analyses of a number of postnatal tissuesindicated that CDMP-1 could predominantly be detected in newbornarticular and cricoid cartilage. In both cases a single mRNA transcriptof approximately 3 kb was observed. The CDMP-1 mRNA was not detected inpancreas, kidney, skeletal muscle, liver, lung, placenta, brain orheart. In contrast, BMP-3 and BMP-7 transcripts were detected insubsequent hybridizations of the same blots in mRNA samples from lung,kidney, brain and small intestine. This finding was consistent withprevious results disclosed by Vukicevic et al., (J. Histochem. Cytochem.42:869 (1994)). CDMP-2 mRNA was detected in postnatal bovine articularand cricoid cartilage as a 4.6 kb mRNA band. After prolonged exposure,weak hybridization signals were detected at 4.6 kb and 4.0 kb in mRNAfrom colon and small intestine, skeletal muscle and placenta.

[0058] Two other procedures were used to localize and visualizeexpression of the CDMP-1 and CDMP-2 gene products. These approachesrelied on detection of mRNA and protein in tissue sections prepared foranalysis by microscopy.

[0059] Example 7 describes the methods used to demonstrate thepreferential expression of CDMPs during human embryogenesis.

EXAMPLE 7 CDMPs are Preferentially Expressed in the Cartilaginous Coresof Long Bone During Human Embryogenesis

[0060] In Situ Hybridization

[0061] Tissues from human embryos were obtained after pregnancytermination at from 5 to 14 weeks of gestation. Embryo age was estimatedin weeks (W) on the basis of crown-rump length (CRL) and pregnancyrecords of the conceptual age. They were fixed in 4% paraformaldehyde in0.1 M phosphate buffer (pH 7.2), embedded in paraffin, sectionedserially at 5-7 μm, and mounted on silanated slides. The tissues used inthe present study were obtained from legally sanctioned proceduresperformed at the University of Zagreb Medical School. The procedure forobtaining the human autopsy material was approved and controlled by theInternal Review Board of the Ethical Committee at the School ofMedicine, University of Zagreb and Office of Human Subjects Research(OHSR) at the National Institutes of Health, Bethesda, Md. In situhybridization was done as described by Vukicevic et al., (J. Histochem.Cytochem. 42:869 (1994)) and by Pelton et al. (Development 106:759(1989)). Briefly, sections were incubated overnight at 50° C. in ahumidified chamber in 50% formamide, 10% dextran sulfate, 4×SSC, 10 mMdithiothreitol, 1×Denhardt's solution, 500 μg/ml of freshly denaturedsalmon sperm DNA and yeast tRNA with 0.2-0.4 ng/ml ³⁵S labeled riboprobe(1×10⁹ CPM/μg). ApaI fragments of CDMP-1 and of CDMP-2 (described above)from the pro region, subcloned in both sense and anti-sense directioninto pBluescript II (SK)⁺ vector (Strategene, Calif.), were used astranscription templates. Riboprobes were then prepared using T7 RNApolymerase (Sure Site Kit, Novagen, Madison, Wis.) according to themanufacturer's instructions and used with and without prior alkalinehydrolysis. After hybridization, the sections were washed as describedby Lyons et al., in Development 109:833 (1990), to a final stringency of0.1×SSC, 65° C. for 2×15 minutes. After dehydration through a gradedethanol series containing 0.3 M ammonium acetate, slides were coveredwith NTB-2 emulsion (Kodak) and exposed between 1 and 3 weeks. Afterdevelopment, the slides were stained with 0.1% toluidine blue,dehydrated, cleared with xylene and mounted with Permount.

[0062] Immunostaining

[0063] A polyclonal antibody to the peptide QGKRPSKNLKARC (SEQ ID NO:10)(amino acids 388-400; prepared by Peptide Technologies, Gaithersburg,Md.), which belongs to the mature secreted protein of CDMP-1, was raisedin rabbits. Before immunization, the peptide was conjugated to Imject®Malemide Activated Keyhole Limpet Hemocyanin (Pierce, Rockfor, Ill.).Searches performed using the BLAST (Altschul et al., J. Mol. Biol.215:403 (1990)) network service available through the National Centerfor Biotechnology Information indicated that the peptide does not showsequence identity with any known protein or BMP. The embryonic tissuesections were stained as recommended by the manufacturer usingimmunogold as a detection system (Auroprobe L M; Janssen, Belgium) andcounterstained with 0.1% toluidine blue. The primary antibody (crudeantiserum) was used at a concentration of 15 μg/ml in PBS with 0.5%bovine serum albumin (BSA) for 1 hour. In the controls, the primaryantibody was replaced with BSA, normal rabbit serum, or secondaryantibody alone.

[0064] Results indicated that, at 6 weeks of gestation, CDMP-1transcripts were detected in precartilage condensations within thedeveloping limbs. At 7.5-8.5 weeks of gestation, CDMP-1 mRNA expressionwas found in the cartilaginous cores of long bones, including thearticular surfaces. In areas of active cartilage degradation and bonematrix formation, CDMP-1 expression was also detected in hypertrophicchondrocytes. Remarkably, no expression was detected in the axialskeleton and only low mRNA levels were observed in other tissues, suchas distal convoluted tubules of the developing kidney, brain andplacenta. Immunohistochemical staining indicated that CDMP-1 proteincolocalized with the mRNA. However, in addition to the sites oftranscription, the protein was also found in the surroundingcartilaginous matrix and in osteoblast-like cells from the primaryossification centers of long bones.

[0065] Between 9 and 10 weeks of gestation, CDMP-2 expression waspredominantly localized in the more mature and hypertrophic chondrocytesin regions of invasion by blood vessels through the periosteal bonycollar of the developing long bone. Again, as for CDMP-1, nohybridization was detected in the vertebral bodies in the correspondingsections and stages of human embryonic development. Low expressionlevels were detected in the periosteum.

[0066] The expression pattern of CDMP-2 suggests it is involved in theterminal differentiation of chondrocytes (hypertrophic and mineralizing)and at the earliest stages of endochondral bone formation, includingangiogenesis and osteoblast differentiation. In addition, the relativelyhigh levels of expression (detectable in total RNA blots) in postnatalcartilage suggest possible roles in the maintenance and stabilization ofthe cartilage phenotype after birth.

[0067] We have also designed experiments aimed at determining whetherall of the chondrogenic activity contained in cartilage extracts can beattributed to the proteins encoded by the CDMP-1 and CDMP-2 cDNAs. Ourapproach involves the production and use of neutralizing antibodiesusing synthetic peptides or recombinant CDMP-1 and CDMP-2 proteins asimmunogens. Antibodies raised against these peptides or proteins will betested for their ability to deplete cartilage extracts of chondrogenicactivity. If antibodies specific for the recombinant proteins fail todeplete the extracts of cartilage-forming activity, then residualactivity will be due to factors within the extract that are separatefrom proteins encoded by the CDMP-1 and CDMP-2 proteins. Alternatively,if antibodies raised against the peptides or recombinant proteins canremove cartilage-inducing activity from the extracts, this will confirmthat CDMP-1 and/or CDMP-2 must be responsible for the active agentscontained in the extracts.

[0068] Example 8 describes the methods that will be used to raiseantibodies against synthetic peptides and recombinant CDMP-1 and CDMP-2proteins. Antibodies produced in this fashion will be tested for theirability to deplete extracts containing CDMP activity.

EXAMPLE 8 Production and Use of Anti-CDMP Antibodies

[0069] Specific monoclonal and polyclonal antibodies will be raisedagainst peptides designed from the mature protein of the CDMPs.Preferentially, the region between the protein cleavage site and thefirst cysteine of the CDMP-1 and CDMP-2 proteins will be used to designthe peptides. In addition, the cDNAs encoding the mature region of theCDMPs will be subcloned in the bacterial pET expression vector, andexpressed as monomers in the bacterial expression system. The proteinexpressed in this system will be used to raise additional antibodies,and to determine the immunoreactivity of the various antisera in Westernblots. The bacterially expressed monomers will be refolded intobiologically active dimers using standard protocols. This approach mayafford another source of recombinant protein.

[0070] The antisera obtained in this fashion will be used to furtherestablish the synthesis of the CDMPs by chondrocytes in vivo and invitro, and to link the cloned CDMPs to the chondrogenic activity foundin cartilage extracts. Conditioned media obtained from chondrocytecultures and partially purified chondrogenic cartilage extracts afterheparin sepharose affinity chromatography, molecular sievechromatography and Con A chromatography, will be analyzed for thepresence of CDMPs by Western blot analysis. Due to the possibleheterogeneity of the highly purified chondrogenic cartilage extracts,the antibodies will be used to reduce or deplete thechondrogenic/osteogenic activity in purified fractions in a standardimmunoprecipitation experiment.

[0071] An important aspect of our invention regards the production anduse of recombinant proteins that possess the biological activities ofthe CDMPs. The following Example describes methods and results thatillustrate the production of recombinant CDMP-1 and CDMP-2 intransfected 293 cells, COS-1 cells, and CHO-1 cells. We discovered that293 cells express BMP-7 that could conceivably contaminate preparationsof recombinant CDMPs. To avoid possible ambiguities in theinterpretation of our results, recombinant CDMP-1 produced in COS-1cells was used to demonstrate cartilage forming activity. Although theproduction of recombinant CDMPs in this fashion was rather inefficient,the key finding illustrated by our results was that recombinant proteinhad the desired cartilage-forming activity. Unexpectedly, and incontrast to the related BMPs, recombinant CDMP-1 induced cartilageformation without noticeable bone formation.

[0072] Example 9 describes the procedures used to produce recombinantCDMP proteins. The results presented in the Example confirm that therecombinant cartilage-derived proteins stimulated cartilage formation.

EXAMPLE 9 Production of Recombinant CDMPs and Assessment of Bioactivity

[0073] Full length CDMP-1 was subcloned into the mammalian expressionvector pcDNA3 (Invitrogen Corporation, San Diego, Calif.) containing thecytomegalovirus early gene promotor and other elements required forexpression in mammalian cells. COS 1 cells were cultured in Opti-MEM I(Gibco/BRL, Gaithersburg, Md.) in the presence of 5% fetal bovine serumand antibiotics. The cells were grown to approximately 70-80% confluencyin 150 mm dishes and transfections of the respective plasmids (20 μgplamid) were carried out by the calcium phosphate method using thetransfection MBS mammalian transfection kit (Stratagene, La Jolla,Calif.). The cells were incubated with the calcium phosphate-DNA mixturefor 3 hours at 35° C. Supernatants were removed and the plates werewashed 3 times with PBS. 15 ml of Opti-MEM I (serum reduced medium) wasadded in the absence of serum, and the dishes were incubated overnight.Transfection efficiencies were tested by transfection of a controlplasmid, pCMVβ-gal and cell extracts were assayed for β-galactosidaseactivity. Conditioned media were collected at 24 hour intervals for aperiod of 96 hours. The pooled media was centrifuged to remove celldebris and then concentrated using Mascrosep 10 concentrators (FiltronTechnology Inc., Northborough, Mass.). Further purification ofrecombinantly expressed protein was performed as described in precedingExamples. In one exemplary procedure, the conditioned media was adjustedto 4 M urea, 25 mM Tris HCl (pH 7.0) and applied to a heparin Sepharosecolumn. The column was washed with the same buffer containing 0.1 MNaCl, and eluted with 1 M NaCl. The heparin Sepharose unbound and elutedfractions were assayed for biological activity as described by Luyten etal., in J. Biol. Chem. 264:13377 (1989).

[0074] Biological activity of the recombinantly expressed protein wasinvestigated using in vitro and in vivo chondrogenic/osteogenic assays.For the in-vivo assay, fractions containing the CDMPs were precipitatedwith ethanol, or dried onto a carrier such as bone matrix residue(mainly collagen type I particles) and cartilage matrix residue(cartilage tissue after extraction with chaotropic agents, andpowderized to particles with a size of 75-400 μm). The dried pellet(about 25 mg) was implanted subcutaneously in rats. Implants wereharvested after 11 and 21 days, and analyzed forchondrogenesis/osteogenesis using alkaline phosphatase determinations.Histological analysis of recovered samples was also performed usingtoluidine blue, alcian blue and safranin O staining. Results obtainedusing the recombinant CDMP-1 produced in COS-1 cells revealedchondrogenic activity in this in vivo assay. Significantly, noosteogenic activity was observed in any of the recovered samples.Osteogenic activity would ordinarily have been observed if the sameprocedures had been carried out using recombinant BMPs. This differencehighlighted the unique properties of recombinant CDMP-1.

[0075] Future in vitro chondrogenic experiments will be performed todetermine the precursor cells responsive to the CDMPs. Undifferentiated(10T1/2 cells, bone marrow stromal cells, mesenchymal stem cells) andalready committed skeletal cells (limb bud cells, perichondrial orperiosteal cells, fetal epihyseal chondroblasts, and chondrocytes) willbe transfected with the cDNAs or treated with recombinantly expressedCDMPs to evaluate the stage of differentiation associated with thechondrogenic activity of the CDMPs.

[0076] Future in vivo chondrogenic experiments will be directed toexpression of large quantities of CDMP-1 and CDMP-2 by stabletransfectants. We contemplate the use of hybrid expression constructs inwhich the pro-region of one BMP family member (for example BMP-2) isoperationally linked to the regions encoding the mature CDMPs. We alsoanticipate in vivo assays based on implantation in other sites, apartfrom subcutaneous implantation, which may reveal distinct or superiorbiological activities of the CDMPs. For example, we anticipateimplantation in the synovial cavity may have utility in such assays.

[0077] The CDMPs disclosed in the present invention have importantapplications in the repair of cartilage defects. We contemplate twogeneral approaches for this type of therapy. In the first place, theCDMPs are used as lineage-specific growth factors for the ex vivoexpansion of chondrocytes isolated from a donor who requires therapeuticintervention. Following expansion, these cells can be reimplanted into acartilage lesion in the donor, whereafter repair of cartilage will takeplace. In a different scenario, CDMPs are introduced into a cartilagelesion. For example, a composition containing an appropriate CDMP ormixture of CDMPs can be implanted into a lesion for the purpose ofstimulating in vivo chondrogenesis and repair of cartilage. The CDMPscan be combined with any of a number of suitable carriers. Anappropriate carrier can be selected from the group comprising fibringlue, cartilage grafts, and collagens. An implantable mixture can beintroduced into the site of a lesion according to methods familiar tothose having ordinary skill in the art. In one application, wecontemplate that periosteal synovial membrane flap of tissue or inertmaterial can be impregnated with CDMPs and implanted for cartilagerepair.

[0078] Example 10 illustrates one application of the CDMP preparationsdescribed above. Specifically, the following Example describes the useof CDMPs to facilitate repair of cartilage in the knee joint.

EXAMPLE 10 Treatment of Deep Knee Defects With Cartilage-DerivedMorphogenetic Proteins

[0079] A young patient having a large defect in the articular surface ofthe knee joint is identified. According to standard surgical procedures,a periosteal flap is obtained from the bone beneath the joint surface ofrib cartilage. The tissue flap is pre-soaked in an extract containingCDMPs or alternatively in a solution containing recombinant CDMPs. Theperiosteal flap treated in this way is then attached over the lesion inthe articular surface of the knee joint by a sewing procedure, forexample using resolvable material. The joint is then closed. The jointis injected with a solution containing CDMPs dissolved or suspended in apharmacologically acceptable carrier to maintain the chondrogenicprocess. Injection is continued until the monitoring physician indicatesrepair of the cartilage is complete. The patient notices markedly lessjoint pain as the cartilage repair process progresses. Exam byarthroscopy indicates repair of the lesion within several weeksfollowing the initial procedure.

[0080] We also contemplate gene therapy protocols based on expression ofCDMP cDNAs or genomic constructs as a way of facilitating in vivocartilage repair. Diseases such as chondromalacia or osteoarthritis areexamples for which such gene therapy protocols are contemplated. Therapymay be achieved by genetically altering synoviocytes, periosteal cellsor chondrocytes by transfection or infection with recombinant constructsthat direct expression of the CDMPs. Such altered cells can then bereturned to the joint cavity. We contemplate that gene transfer can beaccomplished by retroviral, adenoviral, herpesvirus and adeno associatedvirus vectors.

[0081] Both in vivo and ex vivo approaches are anticipated forcontinuously delivering CDMPs for the purpose of retarding ongoingosteoarthritic processes and for promoting cartilage repair andregeneration. In addition, one might employ inducible promoterconstructs (e.g. under transcriptional control of a dexamethasonepromoter) in gene therapy applications of the present invention. Acombined approach to osteoarthritis therapy may have particularadvantages. For example, CDMP-2 could be continuously expressed tosupport the integrity of the articular surface. An inducible constructcould be employed to express CDMP-1 so that chondrogenesis could beaccelerated at the time of more aggressive destruction.

[0082] The foregoing experimental results and characterization confirmedthe CDMP-1 and CDMP-2 isolates belong to the TGF-β superfamily. Based onthe high percentage identity of their C-terminal domains, CDMP-1 andCDMP-2 can be classified as members of a novel subfamily. AlthoughCDMP-1 and CDMP-2 were identified in two different species (human andbovine), they represent distinct genes since the sequences of theirpro-regions are significantly divergent.

[0083] Several BMPs have now been implicated in early skeletaldevelopment, including BMPs -2, -4, -5, -7 and CDMP-1 (GDF-5). Othermembers, such as BMPs -3, -6, -7 and CDMP-2, may be involved in laterstages of skeletal formation (13, 15). The role of the BMPs in earlydevelopment could be chemotactic, mitogenic or inductive. Their functionin later stages of skeletal development might be promotion ofdifferentiation and maintenance of the established phenotype. Theavailability of mouse strains with null mutations in specific BMPmembers, such as the short-ear mice (Bmp5) and the bp mice (Cdmp1/Gdf5),allows analysis of the specific contributions of the respective membersin each of the stages of skeletal development.

[0084] The absence of expression of both CDMP-1 and CDMP-2 in the axialskeleton has implications for models of skeletal development. Forexample, the bp mice have disturbed limb development but a normal axialskeleton. This is the first evidence that the developmental mechanismsand differentiation pathways of the vertebral bodies are distinct fromthose of the peripheral skeletal elements. Further, this indicates thebasic form and pattern of the skeleton are likely to be determined by anumber of BMP-like signaling molecules.

1 24 26 base pairs nucleic acid single linear cDNA not provided Other3...21 inosine (A) NAME/KEY Other (B) LOCATION 24...24 (D) OTHERINFORMATION A or C or G or T 1 GGNTGGMANG AYTGGATHRT NGCNCC 26 9 aminoacids amino acid single linear peptide not provided Other 3...3 Xaa = Qor N (A) NAME/KEY Other (B) LOCATION 7...7 (D) OTHER INFORMATION Xaa = Ior V 2 Gly Trp Xaa Asp Trp Ile Xaa Ala Pro 1 5 26 base pairs nucleicacid single linear cDNA not provided Other 3...24 inosine 3 GGNTGGWSNGARTGGATHAT NWGNCC 26 9 amino acids amino acid single linear peptide notprovided 4 Gly Trp Ser Glu Trp Ile Ile Ser Pro 1 5 23 base pairs nucleicacid single linear cDNA not provided Other 9...9 A or T or G or C 5ARRGTYTGNA CRATRGCRTG RTT 23 8 amino acids amino acid single linearpeptide not provided 6 Asn His Ala Ile Val Gln Thr Leu 1 5 23 base pairsnucleic acid single linear cDNA not provided Other 3...18 inosine 7CANSCRCANS HNYBNACNAY CAT 23 8 amino acids amino acid single linearpeptide not provided Other 2...2 Xaa = V or I (A) NAME/KEY Other (B)LOCATION 4...4 (D) OTHER INFORMATION Xaa = E or R (A) NAME/KEY Other (B)LOCATION 5...5 (D) OTHER INFORMATION Xaa = G or S or A (A) NAME/KEYOther (B) LOCATION 7...7 (D) OTHER INFORMATION Xaa = G or A 8 Met XaaVal Xaa Xaa Cys Xaa Cys 1 5 4 amino acids amino acid single linearpeptide not provided Other 2...2 Xaa = any aa (A) NAME/KEY Other (B)LOCATION 3...3 (D) OTHER INFORMATION Xaa = any aa (A) NAME/KEY Other (B)LOCATION 4...4 (D) OTHER INFORMATION Xaa = R or A 9 Arg Xaa Xaa Xaa 1 13amino acids amino acid single linear peptide not provided 10 Gln Gly LysArg Pro Ser Lys Asn Leu Lys Ala Arg Cys 1 5 10 2341 base pairs nucleicacid single linear cDNA not provided 11 TCAAGAACGA GTTATTTTCA GCTGCTGACTGGAGACGGTG CACGTCTGGA TACGAGAGCA 60 TTTCCACTAT GGGACTGGAT ACAAACACACACCCGGCAGA CTTCAAGAGT TTCAGACTGA 120 GGAGAAAACC TTTCCCTTCT GCTGCTACTGCTGCTGCCGC TGCTTTTGAA AGTCCACTTC 180 CTTTCATGGT TTTTCCTGCC AAACCAGAGGCACCTTCGCT GCTGCCGCTG TTCTCTTTGG 240 TGTCATTCAG CGGCTGGCCA GAGGATGAGACTCCCCAAAC TCCTCACTTT CTTGCTTTGG 300 TACCTGGCTT GGCTGGACCT GGAATTCATCTGCACTGTGT TGGGTGCCCC TGACTTGGGC 360 CAGAGACCCC AGGGGTCCAG GCCAGGATTGGCCAAAGCAG AGGCCAAGGA GAGGCCCCCC 420 CTGGCCCGGA ACGTCTTCAG GCCAGGGGGTCACAGCTATG GTGGGGGGGC CACCAATGCC 480 AATGCCAGGG CAAAGGGAGG CACCGGGCAGACAGGAGGCC TGACACAGCC CAAGAAGGAT 540 GAACCCAAAA AGCTGCCCCC CAGACCGGGCGGCCCTGAAC CCAAGCCAGG ACACCCTCCC 600 CAAACAAGGC AGGCTACAGC CCGGACTGTGACCCCAAAAG GACAGCTTCC CGGAGGCAAG 660 GCACCCCCAA AAGCAGGATC TGTCCCCAGCTCCTTCCTGC TGAAGAAGGC CAGGGAGCCC 720 GGGCCCCCAC GAGAGCCCAA GGAGCCGTTTCGCCCACCCC CCATCACACC CCACGAGTAC 780 ATGCTCTCGC TGTACAGGAC GCTGTCCGATGCTGACAGAA AGGGAGGCAA CAGCAGCGTG 840 AAGTTGGAGG CTGGCCTGGC CAACACCATCACCAGCTTTA TTGACAAAGG GCAAGATGAC 900 CGAGGTCCCG TGGTCAGGAA GCAGAGGTACGTGTTTGACA TTAGTGCCCT GGAGAAGGAT 960 GGGCTGCTGG GGGCCGAGCT GCGGATCTTGCGGAAGAAGC CCTCGGACAC GGCCAAGCCA 1020 GCGGTCCCCC GGAGCCGGCG GGCTGCCCAGCTGAAGCTGT CCAGCTGCCC CAGCGGCCGG 1080 CAGCCGGCCG CCTTGCTGGA TGTGCGCTCCGTGCCAGGCC TGGACGGATC TGGCTGGGAG 1140 GTGTTCGACA TCTGGAAGCT CTTCCGAAACTTTAAGAACT CGGCCCAGCT GTGCCTGGAG 1200 CTGGAGGCCT GGGAACGGGG CAGGACCGTGGACCTCCGTG GCCTGGGCTT CGACCGCGCC 1260 GCCCGGCAGG TCCACGAGAA GGCCCTGTTCCTGGTGTTTG GCCGCACCAA GAAACGGGAC 1320 CTGTTCTTTA ATGAGATTAA GGCCCGCTCTGGCCAGGACG ATAAGACCGT GTATGAGTAC 1380 CTGTTCAGCC AGCGGCGAAA ACGGCGGGCCCCATCGGCCA CTCGCCAGGG CAAGCGACCC 1440 AGCAAGAACC TTAAGGCTCG CTGCAGTCGGAAGGCACTGC ATGTCAACTT CAAGGACATG 1500 GGCTGGGACG ACTGGATCAT CGCACCCCTTGAGTACGAGG CTTTCCACTG CGAGGGGCTG 1560 TGCGAGTTCC CATTGCGCTC CCACCTGGAGCCCACGAATC ATGCAGTCAT CCAGACCCTG 1620 ATGAACTCGA TGGACCCCGA GTCCACACCACCCACCTGCT GTGTGCCCAC GCGGCTGAGT 1680 CCCATCAGCA TCCTCTTCAT TGACTCTGCCAACAACGTGG TGTATAAGCA GTATGAGGAC 1740 ATGGTCGTGG AGTCGTGTGG CTGCAGGTAGCAGCACTGGC CCTCTGTCTT CCTGGGTGGC 1800 ACATCCCAAG AGCCCCTTCC TGCACTCCTGGAATCACAGA GGGGTCAGGA AGCTGTGGCA 1860 GGAGCATCTA CACAGCTTGG TGAAGGGATTCAATAAGCTT GCTCGCTCTC TGAGTGTGAC 1920 TTGGGCTAAA GGCCCCCTTT TATCCACAAGTTCCCCTGGC TGAGGATTGC TGCCCGTCTG 1980 CTGATGTGAC CAGTGGCAGG CACAGGTCCAGGGAGACAGA CTCTGAATGG GACTGAGTCC 2040 CAGGAAACAG TGCTTTCCGA TGAGACTCAGCCCACCATTT CTCCTCACCT GGGCCTTCTC 2100 AGCCTCTGGA CTCTCCTAAG CACCTCTCAGGAGAGCCACA GGTGCCACTG CCTCCTCAAA 2160 TCACATTTGT GCCTGGTGAC TTCCTGTCCCTGGGACAGTT GAGAAGCTGA CTGGGCAAGA 2220 GTGGGAGAGA AGAGGAGAGG GCTTGGATAGAGTTGAGGAG TGTGAGGCTG TTAGACTGTT 2280 AGATTTAAAT GTATATTGAT GAGATAAAAAGCAAAACTGT GCCTAAAAAA AAAAAAAAAA 2340 A 2341 1308 base pairs nucleicacid single linear cDNA not provided 12 CGAGCGTCCG CCGAGCTGGG CTCCGCCAAGGGAATGCGAA CGCGCAAGGA AGGAAGGATG 60 CCGCGGGCGC CGAGAGAGAA TGCCACGGCCCGGGAGCCCC TGGATCGCCA GGAGCCCCCG 120 CCGAGGCCGC AGGAGGAGCC CCAGCGGCGGCCGCCACAGC AGCCTGAAGC TCGGGAGCCT 180 CCCGGCAGGG GCCCGCGCTT GGTGCCCCACGAGTACATGC TGTCAATCTA CAGGACTTAC 240 TCCATCGCCG AGAAGCTGGG CATCAATGCTAGCTTTTTCC AGTCTTCCAA GTCGGCTAAT 300 ACGATCACTA GCTTTGTAGA CAGGGGACTAGACGATCTCT CGCACACTCC TCTCCGGAGA 360 CAGAAGTATT TGTTTGATGT GTCCACGCTCTCAGACAAAG AAGAGCTGGT GGGCGCGGAC 420 GTGCGGCTGT TTCGCCAGGC GCCCGCTGCCCTGGCGCCGC CGGCGGCCGC TCCGCTTGCA 480 GCTCTTCGCC TGCCAGTCGC CCCTGCTGCTGGAAGCGCGG AGCCTGGACC CGCAGGGGCG 540 CCCCGGCCCG GCTGGGAAGT CTTCGACGTGTGGCGGGGCC TGCGCCCCCA GCCCTGGAAG 600 CAGCTGTGCT TGGAGCTTCG GGCCGCGTGGGGCGGCGAGC CGGGCGCCGC GGAGGACGAG 660 GCGCGCACGC CTGGGCCCCA GCAGCCGCCGCCCCCGGACC TGCGGAGTCT GGGCTTCGGC 720 CGGAGGGTGC GGACCCCCCA GGAGCGCGCCTTGCTCGTCG TGTTCTCCAG GTCCCAGCGC 780 AAGACCCTGT TCGCCGAGAT GCGCGAGCAGCTGGGCTCGG CGACCGAGGT GGTCGGCCCC 840 GGTGGTGGGG CCGAGGGGTC GGGGCCGCCGCCGCCGCCGC CGCCGCCGCC GCCGTCGGGC 900 ACCCCGGACG CTGGGCTCTG GTCGCCCTCGCCTGGCCGGC GGCGGCGCAC GGCCTTCGCC 960 AGCCGCCACG GCAAGCGGCA CGGCAAGAAGTCGAGGCTGC GCTGCAGCAA GAAGCCCCTG 1020 CACGTGAACT TCAAGGAGCT GGGCTGGGACGACTGGATTA TCGCGCCCCT GGAGTACGAG 1080 GCCTACCACT GCGAGGGCGT GTGCGACTTCCCGCTACGCT CGCACCTGGA GCCCACCAAC 1140 CACGCCATCA TCCAGACGCT GATGAACTCCATGGACCCCG GCTCCACCCC GCCCAGCTGC 1200 TGCGTGCCCA CCAAATTGAC TCCCATCAGCATCTTGTACA TCGACGCGGG CAATAATGTG 1260 GTCTACAACG AGTACGAGGA GATGGTGGTGGAGTCGTGCG GCTGCAGG 1308 501 amino acids amino acid single linearpeptide not provided 13 Met Arg Leu Pro Lys Leu Leu Thr Phe Leu Leu TrpTyr Leu Ala Trp 1 5 10 15 Leu Asp Leu Glu Phe Ile Cys Thr Val Leu GlyAla Pro Asp Leu Gly 20 25 30 Gln Arg Pro Gln Gly Ser Arg Pro Gly Leu AlaLys Ala Glu Ala Lys 35 40 45 Glu Arg Pro Pro Leu Ala Arg Asn Val Phe ArgPro Gly Gly His Ser 50 55 60 Tyr Gly Gly Gly Ala Thr Asn Ala Asn Ala ArgAla Lys Gly Gly Thr 65 70 75 80 Gly Gln Thr Gly Gly Leu Thr Gln Pro LysLys Asp Glu Pro Lys Lys 85 90 95 Leu Pro Pro Arg Pro Gly Gly Pro Glu ProLys Arg Gly His Pro Pro 100 105 110 Gln Thr Arg Gln Ala Thr Ala Arg ThrVal Thr Pro Lys Gly Gln Leu 115 120 125 Pro Gly Gly Lys Ala Pro Pro LysAla Gly Ser Val Pro Ser Ser Phe 130 135 140 Leu Leu Lys Lys Ala Arg GluPro Gly Pro Pro Arg Glu Pro Lys Glu 145 150 155 160 Pro Phe Arg Pro ProPro Ile Thr Pro His Glu Tyr Met Leu Ser Leu 165 170 175 Tyr Arg Thr LeuSer Asp Ala Asp Arg Lys Gly Gly Asn Ser Ser Val 180 185 190 Lys Leu GluAla Gly Leu Ala Asn Thr Ile Thr Ser Phe Ile Asp Lys 195 200 205 Gly GlnAsp Asp Arg Gly Pro Val Val Arg Lys Gln Arg Tyr Val Phe 210 215 220 AspIle Ser Ala Leu Glu Lys Asp Gly Leu Leu Gly Ala Glu Leu Arg 225 230 235240 Ile Leu Arg Lys Lys Pro Ser Asp Thr Ala Lys Pro Ala Val Pro Arg 245250 255 Ser Arg Arg Ala Ala Gln Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg260 265 270 Gln Pro Ala Ala Leu Leu Asp Val Arg Ser Val Pro Gly Leu AspGly 275 280 285 Ser Gly Trp Glu Val Phe Asp Ile Trp Lys Leu Phe Arg AsnPhe Lys 290 295 300 Asn Ser Ala Gln Leu Cys Leu Glu Leu Glu Ala Trp GluArg Gly Arg 305 310 315 320 Thr Val Asp Leu Arg Gly Leu Gly Phe Asp ArgAla Ala Arg Gln Val 325 330 335 His Glu Lys Ala Leu Phe Leu Val Phe GlyArg Thr Lys Lys Arg Asp 340 345 350 Leu Phe Phe Asn Glu Ile Lys Ala ArgSer Gly Gln Asp Asp Lys Thr 355 360 365 Val Tyr Glu Tyr Leu Phe Ser GlnArg Arg Lys Arg Arg Ala Pro Ser 370 375 380 Ala Thr Arg Gln Gly Lys ArgPro Ser Lys Asn Leu Lys Ala Arg Cys 385 390 395 400 Ser Arg Lys Ala LeuHis Val Asn Phe Lys Asp Met Gly Trp Asp Asp 405 410 415 Trp Ile Ile AlaPro Leu Glu Tyr Glu Ala Phe Gly Cys Glu Gly Leu 420 425 430 Cys Glu PhePro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Val 435 440 445 Ile GlnThr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro Pro Thr 450 455 460 CysCys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu Phe Ile Asp 465 470 475480 Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu 485490 495 Ser Cys Gly Cys Arg 500 436 amino acids amino acid single linearpeptide not provided 14 Arg Ala Ser Ala Glu Leu Gly Ser Ala Lys Gly MetArg Thr Arg Lys 1 5 10 15 Glu Gly Arg Met Pro Arg Ala Pro Arg Glu AsnAla Thr Ala Arg Glu 20 25 30 Pro Leu Asp Arg Gln Glu Pro Pro Pro Arg ProGln Glu Glu Pro Gln 35 40 45 Arg Arg Pro Pro Gln Gln Pro Glu Ala Arg GluPro Pro Gly Arg Gly 50 55 60 Pro Arg Leu Val Pro His Glu Tyr Met Leu SerIle Tyr Arg Thr Tyr 65 70 75 80 Ser Ile Ala Glu Lys Leu Gly Ile Asn AlaSer Phe Phe Gln Ser Ser 85 90 95 Lys Ser Ala Asn Thr Ile Thr Ser Phe ValAsp Arg Gly Leu Asp Asp 100 105 110 Leu Ser His Thr Pro Leu Arg Arg GlnLys Tyr Leu Phe Asp Val Ser 115 120 125 Thr Leu Ser Asp Lys Glu Glu LeuVal Gly Ala Asp Val Arg Leu Phe 130 135 140 Arg Gln Ala Pro Ala Ala LeuAla Pro Pro Ala Ala Ala Pro Leu Ala 145 150 155 160 Ala Leu Arg Leu ProVal Ala Pro Ala Ala Gly Ser Ala Glu Pro Gly 165 170 175 Pro Ala Gly AlaPro Arg Pro Gly Trp Glu Val Phe Asp Val Trp Arg 180 185 190 Gly Leu ArgPro Gln Pro Trp Lys Gln Leu Cys Leu Glu Leu Arg Ala 195 200 205 Ala TrpGly Gly Glu Pro Gly Ala Ala Glu Asp Glu Ala Arg Thr Pro 210 215 220 GlyPro Gln Gln Pro Pro Pro Pro Asp Leu Arg Ser Leu Gly Phe Gly 225 230 235240 Arg Arg Val Arg Thr Pro Gln Glu Arg Ala Leu Leu Val Val Phe Ser 245250 255 Arg Ser Gln Arg Lys Thr Leu Phe Ala Glu Met Arg Glu Gln Leu Gly260 265 270 Ser Ala Thr Glu Val Val Gly Pro Gly Gly Gly Ala Glu Gly SerGly 275 280 285 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Ser Gly Thr ProAsp Ala 290 295 300 Gly Leu Trp Ser Pro Ser Pro Gly Arg Arg Arg Arg ThrAla Phe Ala 305 310 315 320 Ser Arg His Gly Lys Arg His Gly Lys Lys SerArg Leu Arg Cys Ser 325 330 335 Lys Lys Pro Leu His Val Asn Phe Lys GluLeu Gly Trp Asp Asp Trp 340 345 350 Ile Ile Ala Pro Leu Glu Tyr Glu AlaTyr His Cys Glu Gly Val Cys 355 360 365 Asp Phe Pro Leu Arg Ser His LeuGlu Pro Thr Asn His Ala Ile Ile 370 375 380 Gln Thr Leu Met Asn Ser MetAsp Pro Gly Ser Thr Pro Pro Ser Cys 385 390 395 400 Cys Val Pro Thr LysLeu Thr Pro Ile Ser Ile Leu Tyr Ile Asp Ala 405 410 415 Gly Asn Asn ValVal Tyr Asn Glu Tyr Glu Glu Met Val Val Glu Ser 420 425 430 Cys Gly CysArg 435 31 amino acids amino acid single linear peptide not providedOther 3...3 Xaa = I or M or V (A) NAME/KEY Other (B) LOCATION 7...7 (D)OTHER INFORMATION Xaa = D or E (A) NAME/KEY Other (B) LOCATION 11...11(D) OTHER INFORMATION Xaa = Y or F or H (A) NAME/KEY Other (B) LOCATION16...16 (D) OTHER INFORMATION Xaa = L or V (A) NAME/KEY Other (B)LOCATION 18...18 (D) OTHER INFORMATION Xaa = D or E 15 Trp Ile Xaa AlaPro Leu Xaa Tyr Glu Ala Xaa His Cys Glu Gly Xaa 1 5 10 15 Cys Xaa PhePro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acidsamino acid single linear peptide not provided 16 Trp Ile Ile Ala Pro LeuGlu Tyr Glu Ala His His Cys Ala Gly Val 1 5 10 15 Cys Asp Phe Pro LeuArg Ser His Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids aminoacid single linear peptide not provided 17 Trp Ile Ile Ala Pro Leu GluTyr Glu Ala Phe His Cys Glu Gly Asp 1 5 10 15 Cys Glu Phe Pro Leu ArgSer His Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 18 Trp Ile Ile Ala Pro Leu Glu TyrGlu Ala Tyr His Cys Glu Gly Asp 1 5 10 15 Cys Glu Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 19 Trp Ile Val Ala Pro Leu Asp TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 20 Trp Ile Ile Ala Pro Leu Glu TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 21 Trp Ile Ile Ala Pro Leu Glu TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 22 Trp Ile Ile Ala Pro Leu Glu TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 23 Trp Ile Met Ala Pro Leu Asp TyrGlu Ala Tyr His Cys Glu Gly Asp 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30 31 amino acids amino acidsingle linear peptide not provided 24 Trp Ile Ile Ala Pro Leu Glu TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala 20 25 30

We claim:
 1. A purified cartilage extract that stimulates localcartilage formation when combined with a matrix and implanted into amammal, said extract being produced by a method comprising: (a)obtaining cartilage tissue; (b) homogenizing said cartilage tissue inthe presence of chaotropic agents under conditions that permitseparation of proteins from proteoglycans; (c) separating said proteinsfrom said proteoglycans; and (d) obtaining said proteins.
 2. The extractof claim 1 , wherein said extract is obtained by a method in which step(c) comprises use of a sepharose column.
 3. The extract of claim 1 ,wherein said extract is obtained by a method which additionallycomprises the steps of separating said proteins on a molecular sieve andobtaining those proteins having a molecular weight between 30 kDa and 60kDa.
 4. The extract of claim 1 , wherein said extract is an extract ofarticular cartilage.
 5. The extract of claim 1 , wherein said extract isan extract of epiphyseal cartilage.
 6. A method of preparing a partiallypurified articular cartilage extract having chondrogenic activity,comprising the steps: (a) obtaining cartilage tissue; (b) homogenizingsaid cartilage tissue in the presence of chaotropic agents underconditions that permit separation of proteins from proteoglycans; (c)separating said proteins from said proteoglycans; and (d) obtaining saidproteins.
 7. The method of claim 6 , wherein step (c) comprises use of asepharose column.
 8. The method of claim 7 , wherein step (c) comprisesisolating the proteins that bind heparin Sepharose in the presence of0.15 M NaCl but not in the presence of 1 M NaCl.
 9. The method of claim6 , additionally comprising the steps of separating said proteins on amolecular sieve and obtaining those proteins having a molecular weightbetween 30 kDa and 60 kDa.
 10. An isolated DNA molecule encoding aprotein having chondrogenic activity in vivo but substantially noosteogenic activity in vivo, said molecule having a nucleotide sequencethat can hybridize to a polynucleotide having a nucleotide sequenceselected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12 at55° C. with 0.4×SSC and 0.1% SDS.
 11. The isolated DNA molecule of claim10 , wherein said molecule has a nucleotide sequence selected from thegroup consisting of SEQ ID NO:13 and SEQ ID NO:14.
 12. A recombinantprotein having chondrogenic activity in vivo but substantially noosteogenic activity in vivo, wherein said protein has an amino acidsequence selected from the group consisting of SEQ ID NO:13 and SEQ IDNO:14.
 13. A method of stimulating cartilage formation in a mammal,comprising the steps: a) supplying cartilage-derived morphogeneticproteins having in vivo chondrogenic activity; b) mixing said partiallypurified proteins with a matrix to produce a product that facilitatesadministration of said partially purified proteins; and c) implantingthe product of step (b) into the body of mammal to stimulate cartilageformation at the site of implantation.
 14. The method of claim 13 ,wherein said partially purified cartilage-derived morphogenetic proteinsare obtained from articular cartilage or epiphyseal cartilage.
 15. Themethod of claim 13 , wherein the matrix of step (b) comprises a cellularmaterial.
 16. The method of claim 13 , wherein mixing step (b)additionally comprises mixing of viable chondroblast or chondrocytes.17. The method of claim 5 , wherein the implanting step comprisesimplanting subcutaneously.
 18. The method of claim 5 , wherein theimplanting step compises implanting subcutaneously.
 19. The method ofclaim 5 , wherein the implanting step comprises implantingintramuscularly.
 20. A composition that can be administered to a mammalfor the purpose of stimulating chondrogenic activity at the site ofadministration without substantially stimulating osteogenic activity,said composition comprising at least one cartilage-derived morphogeneticprotein and a matrix.
 21. The composition of claim 20 , wherein saidcartilage-derived morphogenetic protein is derived from an extract ofcartilage tissue.
 22. The composition of claim 20 , wherein saidcartilage tissue is selected from the group consisting of articularcartilage and epiphyseal cartilage.
 23. The composition of claim 20 ,wherein said cartilage-derived morphogenetic protein is a recombinantprotein.
 24. The composition of claim 20 , wherein said recombinantprotein has a sequence selected from the group consisting of SEQ IDNO:13 and SEQ ID NO:14.
 25. The composition of claim 20 , wherein saidmatrix is selected from the group consisting of fibrin glue,freeze-dried cartilage, collagens and guanidinium-insoluble collagenousresidue of demineralized bone.
 26. The composition of claim 20 , whereinsaid matrix is a non-resorbable matrix selected from the groupcomprising tetracalcium phosphate and hydroxyapatite.